Our objective is to identify and functionally characterize the genes and genetic pathways underpinning metazoan regeneration. We propose to use the planarian Schmidtea mediterranea as a model system to address this problem because it is among the simplest cephalized bilaterians with complex organ systems that display extensive regenerative capacities: a decapitated animal will regenerate and functionally integrate a new head to the pre-existing tissues in under a week. In addition, such remarkable developmental plasticity is driven by an abundant and experimentally accessible population of totipotent stem cells known as neoblasts. The advances made during the last period of funding to interrogate gene function and measure regenerative responses, combined with the sequencing and recent annotation of the S. mediterranea genome create an unprecedented opportunity to frame the problem of animal regeneration in molecular and functional genomic terms. We propose to take full advantage of the throughput capacity and biological plasticity afforded by planarians to establish a detailed molecular landscape of regeneration. We propose to: 1) carry out genome- wide, high temporal resolution analyses of regeneration using high density DNA arrays;2) test the functions of key, evolutionarily conserved embryonic signaling pathways in the adult contexts of tissue homeostasis and regeneration to provide a functional basis for interpreting the microarray experiments;and 3) to carry out screens of organ-specific regeneration to determine the regulatory extent and degree of molecular specialization that exists in general regenerative events. Combined, these studies should provide us with the most comprehensive mechanistic study of regeneration performed to date, and should serve as a platform to formally test the evolutionary divergence or convergence of regeneration among animals, a central unresolved aspect hindering the implementation of rational regenerative therapeutics in poor regenerators such as mammals. PUBLIC HEALTH RELEVANCE: The overarching goal of this project is to define a mechanistic basis for the process of animal regeneration. This project takes advantage of the sequenced genome of the planarian Schmidtea mediterranea, and of the methodological advances and findings obtained during the last funding period to: 1) define a high temporal resolution, genome- wide, expression profile of regeneration;2) interrogate the functions of known embryonic signaling pathways in the adult contexts of tissue regeneration and homeostasis;and 3) uncover genes involved in the regeneration of adult organs after amputation. All three lines of investigation synergize with each other and their integration should provide us with a high resolution set of molecular processes regulating regeneration and regenerative capacities. In fact, our project has begun to uncover novel functions in adult contexts of known genes, and to define functions for the many conserved animal genes for which functions are still unknown. Given the high degree of evolutionary conservation that exists between planarians and vertebrates, the characterization of gene functions in planarians will advance efforts to study human stem-cell function, regeneration and wound healing, effectively advancing these frontiers of human health.